The mapping of connections among central nervous system (CNS) neurons is a longstanding goal of neuroscientists. Knowledge of the connections will inform our understanding of the function of the nervous system, in both health and disease. Dye labeling, physiological recordings, anatomy at the level of electron microscopy, and the transfer of proteins have all been used to track synaptic connections. Neurotropic viruses have also been used, taking advantage of their ability to spread among neurons. Viral tracers offer the promise of higher throughput, and can expand the repertoire of assays beyond mapping connections, via their gene transfer capabilities. We have recently developed vesicular stomatitis virus (VSV) as a transsynaptic tracer. This virus has several advantages over the previously developed transsynaptic viruses, rabies virus (RABV) and pseudorabies virus (PRV). RABV is a lethal virus, which limits its use in the laboratory. PRV has a large and complex genome, making it difficult to engineer in a straightforward manner. VSV has a relatively simple genome, very similar to that of RABV. It is very well characterized as it rapidly grows to high titr and has a long track record for safety in the laboratory. We found that VSV can transmit transsynaptically in mice, monkeys, and multiple other species, including birds, amphibians, and fish. Moreover, we found that we could create VSV vectors that could transmit specifically retrogradely or specifically anterogradely. The directionality was entirely dependent upon the viral envelope glycoprotein, encoded by a viral G gene. We propose to create a series of transsynaptic viral tracers that will be useful to neuroscientists working in different organisms and different areas of the nervous system. We will first explore the use of VSV with different viral G proteins, applying what we learn to the design of specific anterograde and retrograde tracers. We hope to expand the repertoire of G proteins that can be used not only with VSV, but with other tracers as well. We also propose to develop an alternative non-toxic virus for use as a tracer, using some of the tools that we have already developed for VSV. Finally, we propose to develop a tracer that, although based upon a virus, is actually not a virus, but a vehicle to move Cre recombinase across synapses to map connected neurons. These alternative strategies offer the promise of reduced toxicity, relative to VSV, RABV, or PRV, as well as other features that can be combined with engineered lines of mice, or other model organisms.